Approximately two billion people are colonized with Staphylococcus aureus worldwide, but only a minority of these individuals will develop invasive infections. This application focuses on the role of nitric oxide (NO*), a molecular mediator that modulates bacterial physiology by targeting protein thiols and metal centers, during nasal colonization by S. aureus. We will test the hypothesis that NO* inhibits virulence and regulates microaerobic respiration in S. aureus. Our preliminary observations suggest that NO* is a critical determinant of whether S. aureus exists in a stable commensal relationship with the host or becomes an invasive pathogen. Our specific aims are to: (1) Assess the mechanism of S. aureus virulence gene inhibition by NO* - We have found that NO* inhibits the expression of virulence genes required for invasive infection. Genetic and biochemical approaches will determine how exogenous NO* inhibits virulence gene expression, focusing on AgrA, MgrA, SarR and SarS, four central transcriptional regulators that are S- nitrosylated by NO*. A novel murine nasal colonization model will be used to determine the effects of host-derived NO* on S. aureus virulence gene expression in vivo. (2) Analyze the regulation of S. aureus microaerobic physiology and colonization by NO* - S. aureus produces its own NO* by expressing an enzyme (saNOS) related to mammalian NO* synthases. We have discovered that saNOS is required for redox sensing during the microaerobic transition from aerobic respiration to nitrate respiration and for nasal colonization in mice. This aim will use expression and biochemical assays to test a novel mechanistic model in which bacterial-derived NO* diverts electron transport to nitrate reductase when O2 concentrations are limiting. Our mouse model will be used to assess the contribution of host- and bacterial-derived NO* to S. aureus colonization in vivo. These studies will establish NO* as a critically important signaling molecule that allows S. aureus to adapt to oxygen-limited conditions within the host environment while maintaining it in a commensal state by modulating virulence gene expression.